This invention relates to crosslinked hydrogel networks that include the hydrophilic polymer poly(ethylene glycol).
Chemical attachment of the hydrophilic polymer poly(ethylene glycol )(PEG), also known as polylethylene oxide) (PEO), to molecules and surfaces is of great utility in biotechnology. In its most common form, PEG is a linear polymer terminated at each end with hydroxyl groups:
HOxe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH
This polymer can be represented in brief form as HOxe2x80x94PEGxe2x80x94OH where it is understood that the xe2x80x94PEGxe2x80x94 symbol represents the following structural unit:
xe2x80x94CH2CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94
In typical form, n ranges from approximately 10 to approximately 2000.
PEG is commonly used as methoxy-PEGxe2x80x94OH, or mPEG in brief, in which one terminus is the relatively inert methoxy group, while the other terminus is a hydroxyl group that is subject to ready chemical modification.
CH3Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OHmPEG
PEG is also commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, pentaerythritol and sorbitol. For example, the four-arm, branched PEG prepared from pentaerythritol is shown below;
C(CH2xe2x80x94OH)4+nC2H4Oxe2x86x92C[CH2Oxe2x80x94(CH2CH2O)nxe2x80x94CH2CH2xe2x80x94OH]4
The branched PEGs can be represented in general form as R(xe2x80x94PEGxe2x80x94OH)n in which R represents the central xe2x80x9ccorexe2x80x9d molecule, such as glycerol or pentaerythritol, and n represents the number of arms.
PEG is a much used polymer having the properties of solubility in water and in many organic solvents, lack of toxicity,land lack of immunogenicity. One use of PEG is to covalently attach the polymer to insoluble molecules to make the resulting PEG-molecule xe2x80x9cconjugatexe2x80x9d soluble. For example, Greenwald, Pendri and Bolikal in J. Org. Chem., 60, 331-336 (1995) have shown that the water-insoluble drug taxol, when coupled to PEG, becomes water soluble.
Davis et al. U.S. Pat. No. 4,179,337 describes proteins coupled to PEG and having enhanced blood circulation lifetime because of reduced rate of kidney clearance and reduced immunogenicity. The lack of toxicity of the polymer and its rapid clearance from the body are advantageous features for pharmaceutical applications. These applications and many leading references are described in the book by Harris (J. M. Harris, Ed., xe2x80x9cBiomedical and Biotechnical Applications of Polyethylene Glycol Chemistry,xe2x80x9d Plenum, New York, 1992).
To couple PEG to a molecule such as a protein it is necessary to use an xe2x80x9cactivated derivativexe2x80x9d of the PEG having a functional group at the terminus suitable for reacting with some group on the surface or on the protein (such as an amino group). Among the many useful activated derivatives of PEG is the succinimidyl xe2x80x9cactive esterxe2x80x9d of carboxymethylated PEG as disclosed by K. Iwasaki and Y. Iwashita in U. S. Pat. No. 4,670,417. This chemistry is illustrated with the active ester reacting with amino groups of a protein (the succinimidyl group is represented as NHS and the protein is represented as PRO-NH2):
PEGxe2x80x94Oxe2x80x94CH2xe2x80x94CO2xe2x80x94NHS+PRO-NH2xe2x86x92PEGxe2x80x94Oxe2x80x94CH2xe2x80x94CO2xe2x80x94NH-PRO
Succinimidyl xe2x80x9cactive estersxe2x80x9d, such as PEGxe2x80x94Oxe2x80x94CH2xe2x80x94CO2xe2x80x94NHS, are commonly used forms of activated carboxylic acids, and they are prepared by reacting carboxylic acids with N-hydroxylsuccinimide.
Problems have arisen in the art. Some of the functional groups that have been used to activate PEG can result in toxic or otherwise undesirable residues when used for in vivo drug delivery. Some of the linkages that have been devised to attach functional groups to PEG can result in an undesirable immune response. Some of the functional groups do not have sufficient or otherwise appropriate selectivity for reacting with particular groups on proteins and can tend to deactivate the proteins.
PEG hydrogels, which are water-swollen gels, have been used for wound covering and drug delivery. PEG hydrogels are prepared by incorporating the soluble, hydrophilic polymer into a chemically crosslinked network or matrix so that addition of water produces an insoluble, swollen gel. Substances useful as drugs typically are not covalently attached to the PEG hydrogel for in vivo delivery. Instead, the substances are trapped within the crosslinked matrix and pass through the interstices in the matrix. The insoluble matrix can remain in the body indefinitely and control of the release of the drug can be somewhat imprecise.
One approach to preparation of these hydrogels is described in Embrey and Graham""s U.S. Pat. No. 4,894,238, in which the ends of the linear polymer are connected by various strong, nondegradable chemical linkages. For example, linear PEG can be incorporated into a crosslinked network by reacting with a triol and a diisocyanate to form hydrolytically-stable (xe2x80x9cnondegradablexe2x80x9d) urethane linkages.
A related approach for preparation of nondegradable PEG hydrogels Las been demonstrated by Gayet and Fortier in J. Controlled Release, 38, 177-184 (1996) in which linear PEG was activated as the p-nitrophenylcarbonate and crosslinked by reaction with a protein, bovine serum albumin. The linkages formed are hydrolytically-stable urethane groups.
N. S. Chu U.S. Pat. No. 3,963,805 describes nondegradable PEG networks have been prepared by random entanglement of PEG chains with other polymers formed by use of free radical initiators mixed with multifunctional monomers. P. A. King U.S. Pat. No. 3,419,006 describes the preparation of nondegradable PEG hydrogels by radiation-induced crosslinking of high molecular weight PEG.
Nagaoka et al. U.S. Pat. No. 4,424,311 describes PEG hydrogels prepared by copolymerization of PEG methacrylate with other comonomers such as methyl methacrylate. This vinyl polymerization will produce a polyethylene backbone with PEG attached. The methyl methacrylate comonomer is added to give the gel additional physical strength
Sawhney, Pathak and Hubbell in Macromolecules, 26, 581 (1993) describe the preparation of block copolymers of polyglycolide or polylactide and PEG that are terminated with acrylate groups, as shown below:
CH2xe2x95x90CHxe2x80x94COxe2x80x94(Oxe2x80x94CH2xe2x80x94CO)nxe2x80x94PEGxe2x80x94(Oxe2x80x94CH2xe2x80x94CO)nxe2x80x94Oxe2x80x94COxe2x80x94CHxe2x95x90CH2
In the above formula, the glycolide blocks are the xe2x80x94Oxe2x80x94CH2xe2x80x94COxe2x80x94 units; addition of a methyl group to the methylene gives a lactide block; n can be multiples of 2. Vinyl polymerization of the acrylate groups produces an insoluble, crosslinked gel with a polyethylene backbone. The polylactide or polyglycolide segments of the polymer backbone, being ester groups, are susceptible to slow hydrolytic breakdown, with the result that the crosslinked gel undergoes slow degradation and dissolution.
Substantial non-PEG elements are introduced into the hydrogel. Non-PEG elements tend to introduce complexity into the hydrogelland degradation and dissolution of the matrix can result in undesirable or toxic components being released into the blood stream when the hydrogels are used in vivo for drug delivery.
It would be desirable to provide alternative PEG hydrogels that are suitable for drug delivery and that have unique properties that could enhance drug delivery systems.
The invention provides chemically crosslinked PEG hydrogels for controlled release of conjugates of PEG and various molecules, including, for example, conjugates of PEG and enzyme""s, polypeptides, drugs, nucleosides, phospholipids, and other bioactive substances. The invention also provides methods for preparing the hydrogels.
The hydrogels of the invention are formed by reaction of active derivatives of poly(ethylene glycol) with amine groups on the bioactive substance or other molecule and with amine groups on other poly(ethylene glycol) molecules or related similar nonpeptidic polymers that typically do not contain hydrolytically unstable linkages. The polyethylene glycol) molecules that contain weak linkages in their backbones permit hydrolytic degradation of the crosslinks in the polymer matrix and release of the biloactive substance with the other poly(ethylene glycol) or related nonpeptidic polymer attached. Degradation of the gel in vivo releases PEG/molecule conjugates into the blood stream and produces substantially nontoxic polymer fragments that typically are cleared from the body. Variation of the atoms near the hydrolytically unstable linkages can provide precise control of hydrolytic breakdown rate and release of the conjugate.
Examples of hydrolytically unstable linkages in the PEG polymer backbone include carboxylate ester, phosphate ester, acetals, imines, orthoesters, peptides, anhydrides, ketals, and oligonucleotides. These weak links are formed:by reaction of two PEGs having different terminal groups as illustrated below:
xe2x80x94PEGxe2x80x94Z+Yxe2x80x94PEGxe2x80x94xe2x86x92xe2x80x94PEGxe2x80x94Wxe2x80x94PEGxe2x80x94
In the above illustration, xe2x80x94Wxe2x80x94 represents the hydrolytically unstable weak link. Zxe2x80x94 and Yxe2x80x94 represent groups located at the terminus of the PEG molecule that are capable of reacting with each other to form weak links xe2x80x94Wxe2x80x94. Examples of pairs of Z and Y groups that react to form hydrolytically unstable linkages W include pairs selected from the group consisting of alcohol, and carboxylic acid reacting to form carboxylate esters, amine and aldehyde reacting to form imines, hydrazide and aldehyde reacting to form hydrozones, alcohol and phosphate reacting to form phosphate ester, aldehyde and alcohol reacting to form acetals, alcohols and formats reacting to form orthoesters, peptides formed by the reaction of PEG amine with PEG-peptide terminated with carboxyl to form a new peptide linkage, peptides formed by the reaction of PEG carboxylic acid with PEG-peptide terminated wit amine to form a new peptide linkage, and oligonucleotides formed by reaction of PEG phosphoramidite with an 5xe2x80x2-hydroxyl-terminated PEG oligonucleotide.
For example, the following pairs of Z and Y groups can be used to form some of the W groups described above:
xe2x80x94PEGxe2x80x94CO2H+HOxe2x80x94PEGxe2x80x94xe2x86x92xe2x80x94PEGxe2x80x94CO2xe2x80x94PEGxe2x80x94ester
xe2x80x94PEGxe2x80x94OPO3H2+HOxe2x80x94PEGxe2x86x92xe2x80x94PEGxe2x80x94OPO3(H)xe2x80x94PEGxe2x80x94phosphate ester
xe2x80x94PEGxe2x80x94CHO+(HOxe2x80x94PEG)2xe2x80x94xe2x86x92xe2x80x94PEGxe2x80x94CH(Oxe2x80x94PEG)2xe2x80x94acetal
xe2x80x94PEGxe2x80x94CHO+NH2xe2x80x94PEGxe2x80x94xe2x86x92xe2x80x94PEGxe2x80x94CHxe2x95x90Nxe2x80x94PEGxe2x80x94imine
The PEG hydrogels gels are prepared by mixing three ingredients: (1) a PEG with hydrolytically unstable linkages W in the backbone and with reactive groups X at the ends of the chain, (2) a branched PEG or related nonpeptidic polymer with reactive groups Q at the ends of the chain, and (3) a bioactive molecule or other molecule containing reactive groups Q. Reactive groups X are select ed from the group consisting of succinimidyl (NHS), as in xe2x80x94Oxe2x80x94(CH2)nxe2x80x94CO2xe2x80x94NHS or xe2x80x94Oxe2x80x94CO2xe2x80x94NHS, and related activating groups, including sulfosuccinimidyl, benzotriazole, and p-nitophenyl. Reactive groulps Q typically are amine, xe2x80x94NH2.
A crosslinked network is produced that is held together by hydrolytically unstable groups W and groups T, which are hydrolytically stable. Hydrolysis of the unstable groups W releases the bioactive or other molecule with PEG or a related polymer attached, usually by a covalent linkage., which is hydrolytically stable.
The degree of branching of the polymers can be varied in the hydrogels of this invention to control the physical strength and compressibility of the gels. In general, the greater the degree of branching and the shorter the branches, the greater the strength of the gels, the smaller the pores,land the lower the water content. Strength in this context is defined as resistance to compression or stretching.
The rate of release:e of molecules trapped within the hydrogel matrix is controlled by controlling the hydrolytic breakdown rate of the gel. The hydrolytic breakdown rate oft, the gel can be adjusted by controlling the degree of bonding of the PEGs that form the hydrogel matrix. A multiarm PEG having 10 branches or arms will break down and release drug molecules more slowly than a 3 arm PEG.
The following PEG has been made with two hydrolytically unstable ester linkages in its backbone:
NHSxe2x80x94O2Cxe2x80x94CH2xe2x80x94Oxe2x80x94PEGxe2x80x94Oxe2x80x94CH2xe2x80x94CO2xe2x80x94PEGxe2x80x94O2Cxe2x80x94CH2xe2x80x94Oxe2x80x94PEGxe2x80x94Oxe2x80x94CH2xe2x80x94CO2xe2x80x94NHS
The above PEG is activated at each terminus with an N-hydroxylsuccinimide moiety (NHS) in which the active succinimidyl ester moiety is NHSxe2x80x94CO2xe2x80x94 and is reactive with amino groups. A crosslinked network is produced that is held together by stable amide linkages and by hydrolytically unstable ester linkages when the above molecule is coupled with a multiarm PEG amine and with, for example, a protein that contains additional amino groups. The stable amide linkages are formed from reaction of the active NHS ester with amine.
The above example illustrates some of the advantageous features of the invention. First, the crosslinked network degrades, or breaks down because of hydrolysis of the hydrolytically unstable ester linkages (W) in the PEG backbone. Second, when the gel breaks down, it releases PEG and protein conjugates, potentially useful for therapeutic application. Third, subtle variation of the ester linkage provides control over the hydrolytic breakdown rate.
In the above example the ester linkage has the following structure:
xe2x80x94PEGxe2x80x94Oxe2x80x94CH2xe2x80x94CO2xe2x80x94PEGxe2x80x94
This ester group will hydrolyze with a half life of 4 days at pH 7 and 37xc2x0 C. However, if an ester with the following structure is used, then the half life of hydrolytic degradation of the ester linkages is 43 days at pH 7 and 37xc2x0 C.
xe2x80x94PEGxe2x80x94Oxe2x80x94(CH2)nxe2x80x94CO2xe2x80x94PEGxe2x80x94n=2
Thus, by controlling the identity of the atoms adjacent to the ester linkage it is possible to vary the hydrolytic breakdown rate of the gel. Hence, it is possible to control the rate of release of PEG and protein conjugates bound within the matrix. In general, increasing the n value, which is the number of methylene groups in the above structure, decreases the hydrolysis rate.
Thus, the invention provides, among other things, degradable PEG hydrogels having hydrolytically unstable linkages in which the rate of hydrolysis of the unstable linkages can be controlled for release into the blood stream of conjugates of PEG or related nonpeptidic polymers and proteins or other molecules having some therapeutic effect.
The foregoing and other objects of the invention, and the manner in which the same are accomplished, will be more readily apparent upon consideration of the following detailed description of the invention taken in conjuction with the accompanying drawing, which illustrates an exemplary embodiment.